WO1994003500A1 - Glucans with immunostimulant activity - Google Patents

Glucans with immunostimulant activity Download PDF

Info

Publication number
WO1994003500A1
WO1994003500A1 PCT/EP1993/002063 EP9302063W WO9403500A1 WO 1994003500 A1 WO1994003500 A1 WO 1994003500A1 EP 9302063 W EP9302063 W EP 9302063W WO 9403500 A1 WO9403500 A1 WO 9403500A1
Authority
WO
WIPO (PCT)
Prior art keywords
glucans
glucan
activity
obtainable
mouse
Prior art date
Application number
PCT/EP1993/002063
Other languages
French (fr)
Inventor
Antonio Cassone
Francesco Bistoni
Pier Francesco Marconi
Roberto Germogli
Original Assignee
Consiglio Nazionale Delle Ricerche
Istituto Superiore Di Sanitá
Consulfram S.R.L.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Consiglio Nazionale Delle Ricerche, Istituto Superiore Di Sanitá, Consulfram S.R.L. filed Critical Consiglio Nazionale Delle Ricerche
Priority to AU47063/93A priority Critical patent/AU4706393A/en
Priority to EP93917731A priority patent/EP0654047A1/en
Publication of WO1994003500A1 publication Critical patent/WO1994003500A1/en

Links

Classifications

    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00Medicinal preparations containing organic active ingredients
    • A61K31/70Carbohydrates; Sugars; Derivatives thereof
    • A61K31/715Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • CCHEMISTRY; METALLURGY
    • C08ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08BPOLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • C08B37/0006Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid
    • C08B37/0024Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid beta-D-Glucans; (beta-1,3)-D-Glucans, e.g. paramylon, coriolan, sclerotan, pachyman, callose, scleroglucan, schizophyllan, laminaran, lentinan or curdlan; (beta-1,6)-D-Glucans, e.g. pustulan; (beta-1,4)-D-Glucans; (beta-1,3)(beta-1,4)-D-Glucans, e.g. lichenan; Derivatives thereof
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12PFERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00Preparation of compounds containing saccharide radicals
    • C12P19/04Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Biochemistry (AREA)
  • Molecular Biology (AREA)
  • Wood Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Biotechnology (AREA)
  • Epidemiology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Polymers & Plastics (AREA)
  • Veterinary Medicine (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Microbiology (AREA)
  • Public Health (AREA)
  • Materials Engineering (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)

Abstract

The present invention refers to immunostimulant glucans, to a process for their preparation and to pharmaceutical compositions containing them.

Description

GLUCANS WITH IMMtTNOSTIMPLANT ACTIVITY
The present invention refers to im unostimulant glucans, to a process for their preparation and to pharmaceutical compositions containing them.
Glucan is a polysaccharide occurring in nature in the cell wall of fungine microorganism, particularly of yeasts.
Glucans from different sources, e.g. from different microorganisms, are different one from the other and moreover different extraction processes and treatments to which said microorganisms are subjected, including cultural and maintenance conditions, yield different final products. These differences can be noticed both in the three-dimensional structure of the polysaccharide chain, or in the chemical bonds between glucopyranoside units of said chain, and in the biological activity of the glucans as well as in the presence of substances other than glucan in the crude product with consequently greater or lesser difficulties in the purification. Glucans from Saccharomyces cerevisiae or from Lentinus edodes, both having a branched structure with predominance of β-l,3-glucopyranoside bonds, are known.
Said glucans are particularly studied because of their antitumoral and antibacterial activity (Int. J. Cancer 24, 773-779 (1979); Int. J. Immunopharmacol. Vol. 7 No. 5, 747-751 (1985)). Furthermore, they exhibit an immunomodulator effect both in vivo and in vitro (Rev. icrobiol. Vol. 15, 87-96, 1987) and exert a radioprotective action (Methods and Findings Explt. Pharmacol. Vol. 8, No. 3, 151-155 (1986)).
Glucans have been produced also starting from Candida albicans and the immunomodulator effect thereof has been studied (J. Gen. Microbiol. Vol. 134, 1265-74 (1988)).
EP-A-0416343 (16.08.1990) discloses the preparation of parietal glucanic bodies consisting of at least 90% glucan and partly of chitin, by extraction from the strain of Candida albicans ATCC 20955. The process for the preparation of this product, the biological properties of which are not described and which is in fact described as an intermediate useful for the preparation of final products purified to a degree compatible with the pharmaceutical use, comprises the treatment of the cells in autoclave and subsequent repeated extractions with sodium hydroxide and acetic acid at high temperature.
US patent No. 4992540 (12.02.1991) discloses glucans extracted from Saccharomyces cerevisiae as alimentary additives.
We have now found glucans characterized by particularly high immunostimulant activity and by a remarkable safety thanks to the absence of impurities which are often associated with the similar products until now used.
The glucans of the invention have the following characteristics: ratio between β(l-3) and β(l-6) bonds equal to about 1:1; - chitin content from 3 to 5% by weight; residual protein content lower than 0.3%; absence of mannane; enhancing activity of the in vitro NK cytotoxic activity.
The glucans of the invention are obtainable by different yeast species.
Although the use of Candida albicans ATCC 20955, disclosed in EP 0416343^ is preferred, the glucans of the inventions may be obtained from a number of different strains of Candida, Saccharomyces or other yeast or mycetes species.
The extraction process of the glucans of the invention from the cells comprises the following steps: a) culture of the microorganism in liquid medium, with low glucose content; b) treatment of the cell mass in autoclave at temperatures higher than 100°C; c) repeated extractions with sodium hydroxide and diluted organic acid; d) treatment of the extract with detergent at high temperature.
While steps a)-c) are substantially similar to that described in EP0416343, the step d) has never been described and contributes to the peculiar characteristics of the glucans of the invention. These characteristics particularly comprise high immunostimulant activity, higher than that of known glucans, low toxicity and immunogenic activity.
The treatment with detergent at high temperature is typically carried out using sodium 1-5% dodecyl sulfate, preferably about 2%, in a suitable buffer, from 1-3 hours at the boiling temperature. The step b) is preferably carried out at the temperature of about 120°C for 3 hours.
The alternate extractions with NaOH and organic acids such as acetic acid are carried out at temperatures ranging from 80 to 100°C for 24 hours.
Each extraction is followed by washes with water up to neutrality. The alternate extractions with sodium hydroxide and acetic acid are preferably repeated twice, so as to provide an optimal removal of the parietal alkali-soluble glucan and of all the cellular components.
According to a preferred embodiment of the invention, the glucans are preparared starting from Candida albicans strain ATCC 20955. This strain, univocally identified by means of the restriction polymorphism analysis of the cell DNA, has been deposited by the applicants on August 4, 1989 at the American Type Culture Collection according to the Budapest Treaty. As it is known, the safest and most modern method to identify the biotype under exam is the cell DNA restriction polymorphism analysis (DNA restriction fragment lenght polymorphism; Magee et al. Mol. Cell. Biol. 8, 4721, 1988).
The restriction pattern of the strain provides a genetic fingerprint of - ne microorganism and turns out to be different from th c. of all the other men:, ers of the Candida genus.
The cultural, biochemical and biological characteristics of the strain are reported hereinbelow: Cultural characteristics: formation of chlamydospore on corn- meal agar.
Biochemical characteristics: it ferments glucose and maltose with production of acid and gas, saccharose with the production of acid only and it does not ferment lactose. Biological characteristics: it is pathogenic for rabbit and mouse.
The Candida albicans strain ATCC 20955 is kept on Sabouraud agar Difco in refrigerator at 4°C after 24 h growth at 28°C. For the production, the yeast is grown on a medium having a low glucose content so as to favour the production of the cell-wall, e.g. Winge medium, containing glucose and yeast extract, at 28βC for 18-24 hours, monitoring the culture and checking for the presence of the yeast phase only, so as to obtain an optimal glucose-chitin ratio of approximately 20:1.
The cells grown in the culture broth are collected by centrifugation, washed three times with sterile distilled water and suspended again (1-2% w/v) in pH 5 citrate buffer and then placed in autoclave at 121°C for 3 hours so as to cause the rupture of the cells, the solubilization of the fraction consisting of mannan, proteins, mannoproteins and the release of most cell components.
The mass is collected by centrifugation, resuspended (l%-2% w/v) in 1% sterile NaOH and heated to 100°C for 24 hours. The mass is then washed three times with sterile distilled water until neutral reaction and then resuspended (1-2% w/v) in sterile 0.5 M acetic acid and treated at 80°C for 24 hours after having being washed three times with sterile distilled water until neutral reaction.
In order to assure an optimal removal of all the protein components which may be dangerous for the therapeutic use, the obtained glucan is further purified by treatment (1-2% w/v suspension) with a 2% sodium dodecylsulfate solution in Tris EDTA mercaptoethanol for 1,5 hours at the boiling temperature.
The product is washed by centrifugation with sterile distilled water until all the detergent is removed. The obtained glucan may also be sterilized in autoclave at 121βC for 30 minutes and finally it is freeze-dried. The obtained product is insoluble in water, methanol, acetone, ethyl ether, diluted acids and alkali, partially soluble in warm 1 M NaOH (0,06%) and soluble in dimethylsulfoxide; it contains 95-97% glucan together with 3-5% of chitin with a protein content lower than 0.3% (usually from 0.1 to 0.3%) and
13 complete absence of mannan: both the IR and C-NMR (75 MHz at 72βC) spectra show that the polymer is a polysaccharide bound with β(1-3)bonds to form long straight chains from which side chains bound to the main chain through β(l-6) bonds originate. The ratio between β(l-3) and β(l-6) bonds is about 1:1. Also this feature influences the biological activity of the drug since it is reported that the im unological properties of the product are evidently modified by changing significantly this proportion (Jong SC et al.. EOS-J Immunol. Immunopharmacol. 11(3), 115, 1991). From the sprectra, the presence of chitin, which is bound to the structure by covalent bond, is also evident (Kogan G. et al.. Biopolymers 27, 1055, 1988). Thin layer or paper chromatography show the presence of glucose and the absence of mannose, whereas the hexoses -titer is 95-97%.
The so obtained glucans are not antigenic and exhibit biological activities which classify them as Biological Response Modifiers. Particularly, studies carried out on mice showed that the administration of the glucans can induce an increase of the anti- infective activity induced by polymorphonucleates leukocytes and activated macrophages both against chronic and acute infection; they also enhance the antitumor activity due to NK cell and activated macrophages; they significantly increase the interleukin production and particularly that of tumor necrosis factor Ot and interleukin 2; they potentiate the antibody response.
The immunoadjuvant activity in the animal by the parenteral route is very high without remarkable side- effects being noticed and also by the oral route the activity is very interesting, above all as far as the activity on the lung alveolar macrophages is concerned, also perfectly tolerated.
The high purity of the glucans of the invention, mainly in relation to the protein content, imparts to the molecule particularly interesting characteristics from the point of view of tolerability: acute and chronic toxicity tests did not show any toxic local or systemic effect ( D5Q > 1000 mg/kg i.p. in the mouse and in the rat an LD5Q >2000 mg/kg p.o. in the mouse and in the rat, no toxic effect after daily administrations repeated for one year with doses up to 400 mg/kg/die or up to 250 mg/kg/die i.p.). Moreover no mutagenic, teratogenic, embryotoxic properties or anyhow influencing fertility have been noticed.
The following Example further illustrate the invention.
EXAMPLE Candida albicans strains ATCC 20955, maintained on Sabouraud agar slope Difco (glucose 20%, peptone 10%, agar 1.5%, in distilled water, pH 6.5) where grown to a confluent patina in 1-2 days at 28°C, and conserved at ambient temperature or at 4βC. For production, a loopful of C. albicans agar is inoculated in 100 ml of Winge broth (Difco glucose 0.3%, 0.1% Difco yeast extract in distilled water, pH 6.5). The organism was grown at 28°C, under slight stirring (50 rpm) for 18-24 hours until the stationary growth phase was reached (about 2.8 x 10° cells/ml, corresponding to approx. 14 mg of dry weight/ml).
100 ml of broth culture is used to inoculate 1000 ml of Winge medium that are incubεt.ed as mentioned above.
1000 ml c broth culture previously obtained are used to inoculate 10 1 of Winge medium contained in the fermenter. The yeast was grown at 28°C, slightly stirred to 50 rpm, with a stream of air of 1 1/min. and the pH set on 6.5, until the stationary phase of growth Q was reached (about 2.8-4.5 x 10 cells/ml in 24 h). Control were performed during the growth to verify the presence of yeast cells only.
The cells grown in the broth culture are harvested by low speed centrifugation (3000 rpm, 30 min), washed three times with distilled sterile water and re¬ suspended in citrate pH 5 buffer (223 g of citrate sodium/1 of distilled water) at a concentration of 2-4% and the suspension is autoclaved for 3 hours at 121CC. The mass is harvested for centrifugation, re¬ suspended in 1% sterile NaOH at a concentration of about 2-4% and treated for 24 hours in an oil bath at 100βC. The mass is then washed three times via centrifugation with sterile distilled water (neutral reaction) and it is harvested by centrifugation (5000 rpm, 30 min), re-suspended in sterile 0.5 M acetic acid at a concentration of about 2-4% and treated for 24 hours in an oil bath at 80°C.
The mass is washed again three times by centrifugation with sterile distilled water (neutral reaction) .
The alternate treatment with sodium hydroxide and acetic acid is repeated twice.
The mass is harvested by centrifugation, re- suspended at a ratio of about 2-4% in a 2% solution of sodium dodecylsulphate in Tris-EDTA-mercaptoethanol buffer (Tris - 0.1 M, EDTA 5 mM, mercaptoethanol 100 mM, pH 6.8) and boiled for 1.5 hours.
The mass is washed three times by centrifugation with sterile distilled water saline. Possible traces of SDS in glucan are extracted by means of solubilization in dimethylsulfoxide (DMSO) and extracted with water.
Approx. 1 g of glucan is suspended in 25 ml of DMSO and stirred at 77°C until dissolution. The solution obtained is slightly stirred for 15' thereby adding 65 ml of distilled H-O. Addition of water provokes precipitation of glucan. The mixture obtained is slightly stirred for 5', after which other 6: ml of distilled water are added. The mixture is then centrifuged for 5' at 3500 rpm, and the supernatant discarded.
More water is added to the precipitate, in small quantity and slightly stirred. The whole procedure is repeated until a total proportion of DMO/H-o = 1/19 is obtained. The whole process is therefore repeated using twice the volume of DMSO/H-O mixture.
The product collected for centrifugation after the last washing is transferred on trays and placed in an oven for 24 hours at 60βC.
The process yield is approx. 1.8-2.2 g of glucan per litre of initial culture broth. The 13C-NMR spectrum of the solubilized product has been recorded in DMSO-d6 with a Bruker AC 300 apparatus at 75 MHz and 70βC. From the integration of the signals at 103 and 86 ppm corresponding to the β(l- 3) bonds and those at 60.7 and 70 ppm corresponding to the β(l-6) bonds a ratio among the two kinds of bonds of about 1:1 is calculated, whereas the known glucans, such as those described in US 4992540, EP 0416343 and in Biopolymers 27: 1055, 1988, the ratio is generally quite different, about 65:35 (βl-3 : βl:6) for the glucan of US 4992540 and 35:65 for that of EP 0416343. The hexose titre, determined according to th method of Dubois et al.. (Anal. Chem. 28, 350, 1956) i 96.4% (96-97% for the glucan described in US 4992540).
One single spot with Rf identical to that o reference glucose is showed in ascending pape chromatography (eluent pyridin-ethyl acetate-wate
2:5:7) of the glucan subjected to total acid hydrolysis in trifluoroacetic acid IM at 100°C for 12 hours.
The protein content according to Lowry is about 0.16% (0.78% in the glucan described in US 4992540).
In the biological assay, carried out according to the method reported in literature (Marconi P. et al.. Infection and Immunity, 50(1): 297-303, 1985) in repeated tests carried out administering 0.1-1 mg of glucan of the invention or obtained according to the method of US 4992540, by i.p. route, 5 days before the in vitro test carried out against the tumor line NK- sensitive YAC-1, using cell suspensions deriving both from the peritoneal exudate and from the spleen, the results obtained in Tables 1 and 2 are obtained, where an immunoadjuvant activity of the glucan of the invention significantly higher than that of the known product is noticed. TABLE 1: NK Activity (LU 10) of the peritoneal exudate of mice treated with the glucan of
Figure imgf000014_0001
the invention (glucan from C. albicans) or with that of the US Patent 4992540 (glucan from S. cerevisiae)
An imal Dose of glucan Dose of glucan Dose of glucan Dose of glucan No . from C . albicans from S. cerevisiae from C. albicans from S. cerevisiae
0 . 001 mg /mouse 0.001 mg/mouse 0.01 mg/mouse 0.01 mg/mouse
1 15 9 79 45 2 11 14 36 48 3 12 11 33 41 t-υ
4 16 11 40 53 5 4 3 19 30 6 20 14 28 18 7 25 25 99 37 8 9 18 22 26
Mean 14.00 13.13 44.50 37,25 d.s. 6.55 6.49 28.82 11.88
Statistic Non-significant differences Non-significant differences Test Wilcoxon test paired data Wilcoxon test paired data
TABLE 1 (continued)
Animal Dose of glucan Dose of glucan Dose of glucan Dose of glucan No. from C. albicans from S. cerevisiae from C. albicans from S. cerevisiae 0.1 mg/mouse 0.1 mg/mouse 1 mg/mouse 1 mg/mouse
1 143 48 212 211 2 377 67 218 62 3 209 86 314 108 4 132 56 123 195 5 130 49 240 154 6 188 59 250 138 7 408 101 461 172 8 84 72 56 80
Mean 208.88 67.25 234.25 140.00 d.s. 119.77 18,56 121.28 53.45
Statistic Significant differences (p<0.05) Significant differences (p<0.05) Test Wilcoxon test paired data Wilcoxon test paired data
TABLE 2: NK Activity (LU 10) of spleen cells of mice treated with the glucan of
Figure imgf000016_0001
the invention (glucan from C. albicans) or with that of the US Patent 4992540 (glucan from S. cerevisiae)
Animal Dose of glucan Dose of glucan Dose of glucan Dose of glucan No. from C. albicans from S. cerevisiae from C. albicans from S. cerevisiae 0.001 mg/mouse 0.001 mg/mouse 0.01 mg/mouse 0.01 mg/mouse
1 576 629 274 2 284 356 267 3 425 718 485 4 223 386 327 5 764 776 1817 6 1077 1272 864 7 642 745 538 8 804 728 1195
Mean 599.38 701.25 720.88 d. s. 285.60
Figure imgf000016_0003
282.35 547.29
Statistic Non-significant differences Non-significant differences
Figure imgf000016_0002
Test Wilcoxon test paired data Wilcoxon test paired data
TABLE 2 (continued)
Animal Dose of glucan Dose of glucan Dose of glucan Dose of glucan No. from C. albicans from S. cerevisiae from C. albicans from S. cerevisiae 0.1 mg/mouse 0.1 mg/mouse 1 mg/mouse 1 mg/mouse
1 741 248 481 2 381 546 409 3 1208 633 802 4 766 543 1073 5 2824 2653 1204 Ul 6 1368 902 1070 7 1184 560 820 8 1268 759 1411
Mean 1217.50 855.50 908.75 d. s . 730.74 750.41
Figure imgf000017_0001
347.32
Statistic Significant differences (p<0.05) Significant differences (p<0.05) Test Wilcoxon test paired data Wilcoxon test paired data
In view of the above reported pharmacological properties and of the favourable pharmacological characteristics, mainly as far as tolerability and absence of hypersensitization and anaphylaxis phenomena are concerned, the glucans of the invention may be used for the treatment of tumoral diseases, bacterial or viral infections or of any condition in which a modulation of the immune system is desired. For this purpose, the glucans will be administered in form of pharmaceutical compositions suited to the oral, parenteral, rectal or topical administration. Examples of these formulations comprise tablets, capsules, sachets, syrups, solutions, vials, creams, gels, sprays and the like. The daily dosage will be determined by physicians according to the pathologies to be treated and to the patient's condition (weight, sex, age). It will be usually comprised between 0.1 and 50 mg/kg/die in one or more administrations.

Claims

1. Glucans having the following characteristics: ratio between β(l-3) and β(l-6) bonds equal to about 1:1; chitin content from 3 to 5% by weight; residual protein content lower than 0.3%; absence of mannan; enhancing activity of the in vitro NK cytotoxic activity.
2. Glucans according to claim 1 obtainable from yeast cells.
3. Glucans according to claim 2 obtainable from Saccharomyces cerevisiae or Candida albicans strains.
4. Glucans according to claim 3 obtainable from Candida albicans ATCC 20995.
5. A process for the preparation of the glucans of claims 1-4 comprising: a) culture of the microorganism in liquid medium, with low glucose content; b) treatment of the cell mass in autoclave at temperatures higher than 100eC; c) repeated extractions with sodium hydroxide and diluted organic acid; d) treatment of the extract with detergent at high temperature.
6. A process according to claim 5, in which the detergent is 1-5% sodium dodecylsulfate.
7. Use of the glucans of claim 1-5 for the preparation of medicaments having immunostimulating activity.
8. Pharmaceutical compositions containing as the active principle the glucans of claims 1-5 in admixture with a suitable carrier.
PCT/EP1993/002063 1992-08-10 1993-08-03 Glucans with immunostimulant activity WO1994003500A1 (en)

Priority Applications (2)

Application Number Priority Date Filing Date Title
AU47063/93A AU4706393A (en) 1992-08-10 1993-08-03 Glucans with immunostimulant activity
EP93917731A EP0654047A1 (en) 1992-08-10 1993-08-03 Glucans with immunostimulant activity

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
ITMI921967A IT1256035B (en) 1992-08-10 1992-08-10 IMMUNOSTIMULATING ACTIVITY GLUCANS
ITMI92A001967 1992-08-10

Publications (1)

Publication Number Publication Date
WO1994003500A1 true WO1994003500A1 (en) 1994-02-17

Family

ID=11363857

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP1993/002063 WO1994003500A1 (en) 1992-08-10 1993-08-03 Glucans with immunostimulant activity

Country Status (7)

Country Link
EP (1) EP0654047A1 (en)
CN (1) CN1082056A (en)
AU (1) AU4706393A (en)
IT (1) IT1256035B (en)
MX (1) MX9304793A (en)
WO (1) WO1994003500A1 (en)
ZA (1) ZA935730B (en)

Cited By (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2728269A1 (en) * 1994-12-20 1996-06-21 Inst Francais Du Petrole PROCESS FOR TREATING A FERMENTATION MUST CONTAINING POLYSACCHARIDE
US5532223A (en) * 1989-09-08 1996-07-02 Alpha-Beta Technology, Inc. Use of aqueous soluble glucan preparations to stimulate platelet production
US5622939A (en) * 1992-08-21 1997-04-22 Alpha-Beta Technology, Inc. Glucan preparation
US5633369A (en) * 1989-09-08 1997-05-27 Alpha-Beta Technology, Inc. Method for producing soluble glucans
EP0819762A2 (en) * 1996-07-19 1998-01-21 Mibelle AG Cosmetics Process for the isolation of glucan from yeast
US5849720A (en) * 1989-09-08 1998-12-15 Alpha-Beta Technology, Inc. Enhancement of non-specific immune defenses by administration of underivatized, aqueous soluble glucans
US6046323A (en) * 1997-07-29 2000-04-04 The Collaborative Group, Ltd. Conformations of PPG-glucan
US6369216B1 (en) 1998-09-25 2002-04-09 Biopolymer Engineering Pharmaceutical, Inc. Very high molecular weight β-glucans
WO2002012348A3 (en) * 2000-08-03 2002-04-25 Merck Patent Gmbh Isolation of glucan particles and uses thereof
WO2005067977A1 (en) * 2004-01-14 2005-07-28 Pleuran, S.R.O. Method of preparation of a fungal glucane hydrogel having antibacterial and immunostimulant activity, and use thereof
US7022685B2 (en) 1998-09-25 2006-04-04 Biopolymer Engineering, Inc. Very high molecular weight β-glucans
US8563531B2 (en) 2002-08-13 2013-10-22 Biothera, Inc. Methods of using beta glucan as a radioprotective agent
US8883760B2 (en) 2002-09-04 2014-11-11 University Of Louisville Research Foundation, Inc. Cancer therapy using beta glucan and antibodies
WO2022172087A1 (en) * 2021-02-14 2022-08-18 Universidade Catolica Portuguesa-Ucp Yeast glucans, methods and uses thereof

Families Citing this family (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7507724B2 (en) * 2001-01-16 2009-03-24 Sloan-Kettering Institute For Cancer Research Therapy-enhancing glucan
CN101117356B (en) * 2007-09-17 2010-06-09 中国农业大学 Method for preparing water-insoluble beta-1,3/1,6-dextran
CN101885784B (en) * 2010-07-20 2012-08-08 三峡大学 Method for producing glucomannan by liquid culture of konjac cells
CN105907714A (en) * 2016-04-28 2016-08-31 王晓冰 Improved method for cultivating NK (natural killer) cells
WO2020034948A1 (en) * 2018-08-13 2020-02-20 Lifenergy Biotech Corp. Method for in vitro activation of immune cells

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4992540A (en) * 1984-11-28 1991-02-12 Massachusetts Institute Of Technology Glucan composition and process for preparation thereof
EP0416343A2 (en) * 1989-09-04 1991-03-13 Consiglio Nazionale Delle Ricerche Process for preparing a glucane-containing product starting from candida albicans BMM-12
WO1991003495A1 (en) * 1989-09-08 1991-03-21 Alpha Beta Technology, Inc. Method for producing soluble glucans

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4992540A (en) * 1984-11-28 1991-02-12 Massachusetts Institute Of Technology Glucan composition and process for preparation thereof
EP0416343A2 (en) * 1989-09-04 1991-03-13 Consiglio Nazionale Delle Ricerche Process for preparing a glucane-containing product starting from candida albicans BMM-12
WO1991003495A1 (en) * 1989-09-08 1991-03-21 Alpha Beta Technology, Inc. Method for producing soluble glucans

Cited By (25)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5532223A (en) * 1989-09-08 1996-07-02 Alpha-Beta Technology, Inc. Use of aqueous soluble glucan preparations to stimulate platelet production
US5633369A (en) * 1989-09-08 1997-05-27 Alpha-Beta Technology, Inc. Method for producing soluble glucans
US5663324A (en) * 1989-09-08 1997-09-02 Alpha-Beta Technology, Inc. Method for producing underivatized, aqueous soluble β(1-3) glucan
US5849720A (en) * 1989-09-08 1998-12-15 Alpha-Beta Technology, Inc. Enhancement of non-specific immune defenses by administration of underivatized, aqueous soluble glucans
US5811542A (en) * 1989-09-08 1998-09-22 Alpha-Beta Technology, Inc. Method for producing soluble glucans
US5817643A (en) * 1992-08-21 1998-10-06 Alpha-Beta Technology, Inc. Underivatized, aqueous soluable β(1-3) glucan, composition and method of making same
US5622939A (en) * 1992-08-21 1997-04-22 Alpha-Beta Technology, Inc. Glucan preparation
US5783569A (en) * 1992-08-21 1998-07-21 Alpha-Beta Technology, Inc. Uses for underivatized, aqueous soluble β(1-3) glucan and compositions comprising same
FR2728269A1 (en) * 1994-12-20 1996-06-21 Inst Francais Du Petrole PROCESS FOR TREATING A FERMENTATION MUST CONTAINING POLYSACCHARIDE
EP0819762A3 (en) * 1996-07-19 2001-09-05 Mibelle AG Cosmetics Process for the isolation of glucan from yeast
EP0819762A2 (en) * 1996-07-19 1998-01-21 Mibelle AG Cosmetics Process for the isolation of glucan from yeast
DE19629118C2 (en) * 1996-07-19 2001-11-29 Mibelle Ag Cosmetics Buchs Process for isolating glucan from yeast
DE19629118A1 (en) * 1996-07-19 1998-01-22 Mibelle Ag Cosmetics Process for isolating glucan from yeast
US6046323A (en) * 1997-07-29 2000-04-04 The Collaborative Group, Ltd. Conformations of PPG-glucan
US6369216B1 (en) 1998-09-25 2002-04-09 Biopolymer Engineering Pharmaceutical, Inc. Very high molecular weight β-glucans
US7022685B2 (en) 1998-09-25 2006-04-04 Biopolymer Engineering, Inc. Very high molecular weight β-glucans
US7566704B2 (en) 1998-09-25 2009-07-28 Biopolymer Engineering, Inc. Very high molecular weight β-glucans
US9187575B2 (en) 2000-08-03 2015-11-17 Abac R&D Ag Isolation of glucan particles and uses thereof
WO2002012348A3 (en) * 2000-08-03 2002-04-25 Merck Patent Gmbh Isolation of glucan particles and uses thereof
US8563531B2 (en) 2002-08-13 2013-10-22 Biothera, Inc. Methods of using beta glucan as a radioprotective agent
US9522187B2 (en) 2002-09-04 2016-12-20 University Of Louisville Research Foundation, Inc. Cancer therapy using beta glucan and antibodies
US8883760B2 (en) 2002-09-04 2014-11-11 University Of Louisville Research Foundation, Inc. Cancer therapy using beta glucan and antibodies
WO2005067977A1 (en) * 2004-01-14 2005-07-28 Pleuran, S.R.O. Method of preparation of a fungal glucane hydrogel having antibacterial and immunostimulant activity, and use thereof
US7622459B2 (en) 2004-01-14 2009-11-24 Pleuran SRO Method of preparation of a fungal glucane hydrogel having antibacterial and immunostimulant activity, and use thereof
WO2022172087A1 (en) * 2021-02-14 2022-08-18 Universidade Catolica Portuguesa-Ucp Yeast glucans, methods and uses thereof

Also Published As

Publication number Publication date
EP0654047A1 (en) 1995-05-24
ITMI921967A1 (en) 1994-02-10
ZA935730B (en) 1994-03-03
CN1082056A (en) 1994-02-16
IT1256035B (en) 1995-11-21
ITMI921967A0 (en) 1992-08-10
MX9304793A (en) 1994-05-31
AU4706393A (en) 1994-03-03

Similar Documents

Publication Publication Date Title
EP0654047A1 (en) Glucans with immunostimulant activity
JP3651697B2 (en) New polysaccharide substances
Sun et al. Purification, structure and immunobiological activity of a new water-soluble polysaccharide from the mycelium of Polyporus albicans (Imaz.) Teng
López-Legarda et al. Biotechnological production, characterization and in vitro antitumor activity of polysaccharides from a native strain of Lentinus crinitus
CN111234044B (en) Low-molecular-weight tremella aurantialba glucuronic acid-xylan and preparation method and application thereof
KR20100067305A (en) Pharmaceutical composition with antiviral activity extracted from phellinus pini
US20060078971A1 (en) Isoflavone-beta-D-glucan produced by Agaricus blazei in the submerged liquid culture and method of producing same
JP3444624B2 (en) Highly branched β-glucan, its production method and use
US20120124703A1 (en) Novel coprinus comatus and tremella mesenterica mushroom strains, products and extracts thereof and compositions comprising them
KR100197446B1 (en) Anti-cancer immunoactive polysaccharides separated from phellinus linteus and process for the preparation thereof
US4398023A (en) β-1,3-Glucanpolyol, process for preparation thereof, and utilization thereof
US4614733A (en) Polysaccharides pharmaceutical compositions and the use thereof
JP2011522911A (en) Compositions obtained from chlorella extracts having immunomodulatory properties
WO2009017463A2 (en) NOVEL β-GLUCANS ISOLATED FROM HIGHER BASIDIOMYCETES MUSHROOM GANODERMA TSUGAE VAR. JANNIEAE
CN1663959A (en) Beta-glucan peptide and its preparing process and application
Ingólfsdóttir Bioactive compounds from Iceland moss
US6727081B2 (en) Microorganism isolated from chinese elm (Ulmus sp.) and process for preparing exopolysaccharides by employing the microorganism
JPS58318B2 (en) Method for producing anticancer substances
KR20040082060A (en) Composition comprising soluble glucan oligomer from Saccharomyces cerevisiae IS2 for immune activation or prevention and treatment of cancer and the preparation method thereof
CZ466590A3 (en) Extracellular exopolymer, process of its preparation, pharmaceutical composition containing such extracellular exopolymer and use
Blaschek et al. In vitro production of specific polysaccharides: isolation and structure of an antitumor active β-glucan from Phytophthora parasitica
JP4422404B2 (en) Infection prevention / treatment agent and food
KR20050036495A (en) MANUFACTURING METHOD OF SOLUBLE β-GLUCAN
JPH0680703A (en) Water-insoluble polysaccharide originating in mushroom, its production, and antitumor agent mainly comprising the polysaccharide
KR100497751B1 (en) Agrobacterium sp. beta 82 KCTC 10099BP producing water-soluble polysaccharide, water-soluble polysaccharide and producing method thereof

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AT AU BB BG BR CA CH CZ DE DK ES FI GB HU JP KP KR KZ LK LU MG MN MW NL NO NZ PL PT RO RU SD SE SK UA US VN

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE CH DE DK ES FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
WWE Wipo information: entry into national phase

Ref document number: 1993917731

Country of ref document: EP

ENP Entry into the national phase

Ref country code: US

Ref document number: 1995 379660

Date of ref document: 19950320

Kind code of ref document: A

Format of ref document f/p: F

WWP Wipo information: published in national office

Ref document number: 1993917731

Country of ref document: EP

REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

NENP Non-entry into the national phase

Ref country code: CA

WWW Wipo information: withdrawn in national office

Ref document number: 1993917731

Country of ref document: EP